Preparation of stable tau oligomers for cellular and biochemical studies.
Preparation of stable tau oligomers for cellular and biochemical studies.
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DOI：
10.1016/j.ab.2018.10.013
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发表时间：
2019-02-01
影响因子：
2.9
通讯作者：
Moffat KG
中科院分区：
文献类型：
--
作者：
Karikari TK;Nagel DA;Grainger A;Clarke-Bland C;Hill EJ;Moffat KG
越来越多的证据表明，小寡聚体是阿尔茨海默病和其他 tau 病中主要的神经毒性 tau 蛋白。然而，人们对 tau 寡聚体介导的神经变性的机制知之甚少。由于聚集而导致的低聚物的短暂性可能会损害体外制备的低聚物的稳定性。因此，我们寻求开发一种有效的方法来保持预制低聚物的稳定性和球状构象。这项研究表明，在还原条件下用 Alexa Fluor 488-C5-马来酰亚胺或 N-乙基马来酰亚胺标记促聚集 tau 四重复区域 (K18) 的单半胱氨酸形式，可通过阻止其进一步聚集来稳定低聚物。此外，还描述了使用这种方法来研究标记的细胞外 tau K18 寡聚体向人神经母细胞瘤细胞和人干细胞衍生神经元的增殖。该方法可能适用于制备稳定的 tau 寡聚物，用于诊断和生物标志物测试，以及体外结构-活性关系测定。 tau 聚集的瞬时性质使其难以维持预形成的寡聚体的稳定性。使用 Alexa Fluor-488-C5-马来酰亚胺或 N-乙基马来酰亚胺有效标记 tau K18 可稳定所得寡聚物。外源应用的寡聚物通过培养的人 iPSC 神经元和神经母细胞瘤细胞在细胞内繁殖。通过马来酰亚胺标记制备低聚物可以对 tau 聚集及其与神经退行性变的联系进行机制研究。
Increasing evidence suggests that small oligomers are the principal neurotoxic species of tau in Alzheimer's disease and other tauopathies. However, mechanisms of tau oligomer-mediated neurodegeneration are poorly understood. The transience of oligomers due to aggregation can compromise the stability of oligomers prepared in vitro. Consequently, we sought to develop an efficient method which maintains the stability and globular conformation of preformed oligomers. This study demonstrates that labeling a single-cysteine form of the pro-aggregant tau four-repeat region (K18) with either Alexa Fluor 488-C5-maleimide or N-ethylmaleimide in reducing conditions stabilizes oligomers by impeding their further aggregation. Furthermore, the use of this approach to study the propagation of labeled extracellular tau K18 oligomers into human neuroblastoma cells and human stem cell-derived neurons is described. This method is potentially applicable for preparing stabilized oligomers of tau for diagnostic and biomarker tests, as well as for in vitro structure-activity relationship assays. The transient nature of tau aggregation makes it difficult to maintain the stability of preformed oligomers. Efficient labeling of tau K18 with Alexa Fluor-488-C5-maleimide or N-ethyl maleimide stabilizes the resulting oligomers. Oligomers applied exogenously are propagated intracellularly by cultured human iPSC neurons and neuroblastoma cells. Oligomer preparation by maleimide labeling allows mechanistic studies of tau aggregation and its link to neurodegeneration.
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DOI：
10.1074/jbc.m114.554725
发表时间：
2014-07-18
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Kumar S;Tepper K;Kaniyappan S;Biernat J;Wegmann S;Mandelkow EM;Müller DJ;Mandelkow E
通讯作者：
Mandelkow E
影响因子：
6.1
作者：
Combs B;Hamel C;Kanaan NM
通讯作者：
Kanaan NM
影响因子：
15.1
作者：
Lasagna-Reeves CA;Castillo-Carranza DL;Sengupta U;Clos AL;Jackson GR;Kayed R
通讯作者：
Kayed R
DOI：
10.1016/j.bbrc.2011.06.135
发表时间：
2011-07-22
影响因子：
3.1
作者：
Bader, Benedikt;Nuebling, Georg;Giese, Armin
通讯作者：
Giese, Armin
影响因子：
4.8
作者：
Goux, WJ;Kopplin, L;Kirschner, DA
通讯作者：
Kirschner, DA